mAb glycosylation

In article <3A0290D5.2F59107E at shef.ac.uk>, Tom Barr
<URL:mailto:mdp96tab at shef.ac.uk> wrote:
> Joe,
> I think you're right... phage mAbs (scFv) are un-glycosylated and are
> single chains. My mAbs are standard hybridoma produced antibodies. I've
> done a quick test on them and it appears they are glycosylated. I find it
> hard to see how tyhey couldn't be. Howvere, I read in Hermansson
> (Bioconjugate Techniques) that many mAbs are un-glycosylated.... I'm not
> convinced.
>> Cheers,
>> Tom
>
Hi Tom,
I'm not sure what your original question exactly was but regarding antibody
glycosylation here goes.
Antibodies can be glycosylated in both the C-regions and in the V-regions.
Obviously C-region glycosylation is dependent on the particular sequence
which by definition defines the class and subclass of the antibody. Many
classes of antibody have conserved N-linked glycosylation sites in the
constant domains. For example all IgG antibodies have a conserved N-linked
glycosylation site in the CH2 domain at residue Asn297.
Some classes and subclasses also have O-linked sugars, often in the hinge
region, eg IgD and IgA from some species.
V-regions are by definition variable in sequence so sometimes N-linked
glycosylation sites are generated by somatic rearrangement and mutation
(Asn-X-Thr or Asn-X-Ser where X is not Pro). It is estimated that perhaps
as much as 10% of serum Ig is glycosylated in the V-regions.
The next point is that the sugars are added by enzymes called
glycosyl-transferases. Thus the exact structures of sugars added depend
upon which enzymes are expressed in the cell. Different cell types, and
cells from different species will thus glycosylate antibodies differently.
Bacteria do not glycosylate antibodies but plant and insect cells can.
The situation is further complicated in that even in a single cell type
there is not just one N-linked structure added. There will be a complex
mixture of structures such that the population of monoclonal antibody
molecules in a single preparation will complex. This is why the bands
appear on SDS-PAGE as a broad smear rather than a single tight and descrete
band.
All the above information and more can be readily obtained through reading
text books and reviews.
Mike <URL:http://www.path.cam.ac.uk/~mrc7/>
--
o/ \\ // || ,_ o M.R. Clark, PhD. Division of Immunology
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